Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint

EMBO J. 2011 Jul 19;30(16):3322-36. doi: 10.1038/emboj.2011.239.


The spindle assembly checkpoint (SAC) restrains anaphase until all chromosomes become bi-oriented on the mitotic spindle. The SAC protein Mad2 can fold into two distinct conformers, open (O) and closed (C), and can asymmetrically dimerize. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C-Mad2. This antibody revealed several conformation-specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with Mad1 to adopt the closed conformation and that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent 'capping'. Furthermore, C-Mad2 antibody microinjection caused an abrupt termination of the SAC and accelerated mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Our study provides direct in vivo evidence for the model that a kinetochore complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / immunology
  • Adaptor Proteins, Signal Transducing / physiology*
  • Anaphase / physiology*
  • Animals
  • Antibodies, Monoclonal / immunology
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / immunology
  • Calcium-Binding Proteins / physiology*
  • Cdc20 Proteins
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / immunology
  • Cell Cycle Proteins / physiology*
  • Dimerization
  • Humans
  • Kinetochores / metabolism
  • Macromolecular Substances
  • Mad2 Proteins
  • Mice
  • Mice, Inbred BALB C
  • Mitosis / drug effects
  • Mitosis / physiology
  • Nuclear Pore / metabolism
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / immunology
  • Nuclear Proteins / physiology*
  • Protein Conformation
  • Protein Folding
  • Protein Interaction Mapping
  • RNA, Small Interfering / pharmacology
  • Rabbits
  • Repressor Proteins / chemistry
  • Repressor Proteins / immunology
  • Repressor Proteins / physiology*
  • Spindle Apparatus / physiology*
  • Time Factors


  • Adaptor Proteins, Signal Transducing
  • Antibodies, Monoclonal
  • Calcium-Binding Proteins
  • Cdc20 Proteins
  • Cell Cycle Proteins
  • MAD1L1 protein, human
  • MAD2L1 protein, human
  • MAD2L1BP protein, human
  • Macromolecular Substances
  • Mad2 Proteins
  • Nuclear Proteins
  • RNA, Small Interfering
  • Repressor Proteins
  • CDC20 protein, human